A fermentation method for producing substances which involves culturing microorganisms or cultured cells can be roughly classified into (1) a batch fermentation method and a fed-batch or semi-batch fermentation method and (2) a continuous fermentation method. The batch, fed-batch or semi-batch fermentation method has advantages such as use of simple facilities, completion of culture in a short time, and low possibility of contamination with unwanted microorganisms other than cultured ones in product fermentation using pure microorganism culture techniques. However, the concentration of the product in a culture medium increases with the passage of time, leading to reduction in productivity and yield due to inhibition of fermentation by the product or influence of an increase in osmotic pressure. Accordingly, it is difficult to maintain high yield and high productivity stably for long hours.
The continuous fermentation method, on the other hand, can keep a high yield and high productivity for longer hours than the above-mentioned batch, fed-batch or semi-batch fermentation method by preventing accumulation of an objective substance in a fermentor. Conventional continuous culture is a culture method in which a liquid amount in a fermentor is kept constant by feeding the fermentor with a fresh medium while discharging the same amount of the culture medium from the fermentor. In batch culture, culture is terminated when the initial substrate concentration vanishes as a result of consumption, whereas in continuous culture, culture can be theoretically continued infinitely.
In the conventional continuous culture, on the other hand, microorganisms together with a culture medium are discharged from a fermentor so that the concentration of microorganisms in the fermentor is hardly kept high. If the concentration of microorganisms in the fermentor can be kept high, it leads to improvement in the efficiency of fermentation production per fermentation volume. For this purpose, microorganisms should be retained or refluxed in the fermentor.
Examples of the method of retaining or refluxing microorganisms in a fermentor include a method of conducting solid-liquid separation of a discharged culture medium by centrifugal separation and returning precipitated microorganisms to a fermentor and a method of filtering the discharged culture medium to separate microorganisms as solids and discharging only the supernatant of the culture medium from a fermentor. The method using centrifugal separation is however not practical because of a high power cost. The method using filtration requires a high pressure for filtration as described above so that it has been examined mainly at a laboratory level.
There has therefore been proposed a continuous fermentation method to keep the concentration of the microorganisms or cultured cells in a culture medium high. That method includes separating microorganisms or cultured cells through a separation membrane and retaining or refluxing the microorganisms or cultured cells thus separated in a culture medium while recovering a product from the filtrate. For example, there have been disclosed technologies (JP-A-5-95778, JP-A-62-138184 and JP-A-10-174594) relating to membrane separation type continuous fermentation in a continuous fermentation apparatus using a ceramic membrane.
On the other hand, there has recently been proposed a technology of conducting continuous culture by using a continuous culture apparatus using an organic polymer separation membrane (refer to WO 07/097,260 and JP-A-2008-212138). According to that proposal, by using a continuous culture apparatus equipped with a tank for culturing microorganisms or cultured cells and a tank to conduct membrane separation between an intended fermentation product and the microorganisms or cultured cells, a variety of chemicals can be produced at a higher production rate compared with the batch, fed-batch, or semi-batch culture method.
In such continuous fermentation technologies using a separation membrane, reduction in equipment cost, a membrane exchanging cost, and an installation area has been tried by using a separation membrane excellent in water permeability to reduce the area of the membrane, thereby reducing the size of the apparatus from the standpoint of cost reduction. A hollow fiber membrane with a wide filtration area relative to its volume has attracted attentions from such a standpoint of the cost.
Such separation membranes including a hollow fiber membrane sometimes however have deteriorated filtration ability due to SS (Suspended Solids) or adsorbed material attached to the membrane surface during filtrating operation, making it impossible to secure a necessary filtrate amount. With regards to a method of suppressing clogging of the membrane with microorganisms or cultured cells, there have been made several proposals on a technology relating to cleaning of a porous separation membrane or setting of filtering conditions.
As a cleaning method of a porous separation membrane, there have been disclosed, for example, a method of backwashing a porous separation membrane with warm water (JP-A-2000-317273), a method of backwashing a porous separation membrane with a permeate of the filtration (Japanese Patent Laid-Open No. Hei JP-A-11-215980), and the like.
Moreover, it is possible to use a method of scrubbing which conducts cleaning while supplying a gas. Scrubbing cleaning has already been employed for water treatment. For example, Japanese Patent No. 3948593 has proposed a method of introducing a gas in a module and at the same time, introducing a gas or a liquid to the filtrate side of the membrane, thereby cleaning the membrane.
On the other hand, there is an example of using a gas cleaning method in a membrane bioreactor (MBR) for water treatment using high-concentration microorganisms. For example, there is known a method (JP-A-2005-88008) of supplying gas-containing raw water from a raw water supply port provided at the lower portion of a module.
The methods of cleaning a separation membrane described in JP '273 and JP '980 are methods of cleaning a porous separation membrane to be used when a fermentation product is filtered and recovered from a culture medium after completion of fermentation. If such a cleaning method is used for a continuous fermentation method which retains microorganisms or cultured cells in a culture medium after filtration treatment, it is difficult to keep the productivity of fermentation at a high level because the culture medium is diluted.
The technology proposed in JP '593 is a method of treating river surface stream water, used as objective raw water, having a turbidity of from 0.1 to 5. Substances causing clogging are different from those causing clogging during filtration of a culture medium so that this method cannot exhibit its effect fully for suppressing clogging and deterioration in filtration ability in continuous fermentation.
According to JP '008, a gas is supplied under conditions intended to satisfy only the membrane surface cleaning effect and no consideration is given to the influence of an excessively supplied gas on fermentation results and on filtration separation of a product. This means that the technology of JP '008 cannot be applied as is to the production of a chemical.
In the conventional art, an appropriate scrubbing cleaning method for continuous fermentation operation using a membrane separation technology has not been studied. There is therefore a demand for a method of enhancing the fermentation productivity of a chemical while conducting membrane surface cleaning to keep the filterability of a separation membrane.
It could therefore be helpful to provide a method for producing a chemical through continuous fermentation which method requires only a simple and easy operation but keeps high productivity stably for long hours.